Agrobacterium is a genus of soil Gram-negative bacteria that is widely used for the introduction of exogenous DNA into plants. The use of Agrobacterium species for DNA transfer is based on their natural ability to transfer DNA sequences into the genomes of plants. The most widely used species of Agrobacterium is A. tumefaciens the causal agent of the neoplastic disease crown gall in plants. A closely related species, A. rhizogenes, induces hairy root disease and also has been used for DNA transfer to plant genomes, but to a lesser extent. The ability of these bacteria to transfer DNA into plants depends on the presence of large plasmids (>100 kb) within the cells. These plasmids are referred to as the Ti (Tumor inducing) or Ri (Root inducing) in A. tumefaciens and A. rhizogenes, respectively. A third species, A. radiobacter, differs in lacking a Ti or Ri plasmid. The mechanism for DNA transfer from the bacterium into the plant genome involves the mobilization of specific T-DNA (transfer DNA) molecules from the Ti plasmid into the host cell. The T-DNA region is delineated by 25 by referred to as the left and right borders. In pathogenic Agrobacterium cells, within the T-DNA element reside genes for the over production of auxins and cytokinins which manifest the crown gall symptoms. The T-DNA element of pathogenic Agrobacterium strains also contains genes for the production of opines that are utilized by the bacterium as a nitrogen source.
Agrobacterium-mediated DNA transfer to plant cell genomes is usually conducted with “disarmed” (auxin, cytokinin and opine gene sequences removed from the T-DNA element) strains. In transformation studies, sequences of interest are introduced into the T-DNA region of a “disarmed” Agrobacterium strain. This chimeric T-DNA element can be carried on a separate, smaller, wide host range plasmid referred to as a binary vector or directly introduced into the resident “disarmed” Ti plasmid. The first step in the basic Agrobacterium-mediated transformation protocol requires the inoculation of plant cells with transconjugants of “disarmed” Agrobacterium cells carrying the sequences of interest on the chimeric T-DNA element. The plant cells are subsequently cultured for a period generally ranging from one to seven days in a step of the protocol referred to as co-cultivation. Following the co-cultivation period, the plant cells are subcultured on regeneration medium for further plant development.
It is common practice in Agrobacterium transformation processes of plants to use an Agrobacterium counter-selective agent, such as an antibiotic, in the plant culture medium post co-culture as a strategy to counter-select Agrobacterium cells. Counter-selection is used to cure the plant tissue of the Agrobacterium. Without curing, the Agrobacterium would rapidly overgrow and kill the plant tissue, thereby resulting in the production of few or no transgenic events. Furthermore, different strains of Agrobacterium are differently affected by different counter-selective agents, see for example, Ogawa, Y., et al., Screening for highly active beta-lactam antibiotics against Agrobacterium tumefaciens, Arch Microbiol, 181(4):331-6 (2004). Some counter-selective agents have biocidal effects, while others are biostatic. Similarly, plant cell and tissue cultures may have varied responses to bacterial counter-selective agents (for example; Ling, H. Q., et al., Effect of ticarcillin/potassium clavulanate on callus growth and shoot regeneration in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.) Plant Cell Reports, 17:843 (1998); Tang, H., et al., An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants, Plant Cell Reports, 19:881 (2000); Alsheikh, M. K., et al., Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens, Plant Cell Reports, 20:1173 (2002). While some bacterial antibiotics are toxic to plant cell cultures, others may have plant growth regulator activity (for example, Borrelli, G. M., et al., Effect of cefotaxime on callus culture and plant regeneration in durum wheat, Journal of Plant Physiology, 140:372 (1992); Lin, J. J., et al., Plant hormone effect of antibiotics on the transformation efficiency of plant tissues by Agrobacterium tumefaciens cells, Plant Science (Limerick), 109:171 (1995). In the development of any plant transformation protocol via Agrobacterium, it is of importance to use a counter-selective agent that effectively kills the Agrobacterium, but has no undesired effect on the plant tissue (Shackelford, N. J., et al., Identification of antibiotics that are effective in eliminating Agrobacterium tumefaciens, Plant Molecular Biology Reporter, 14:50 (1996). It can be time-consuming and difficult to identify these conditions and one ordinarily skilled in the art must make concessions to achieve an acceptable level of plant transformation efficiency (Ogawa, Y., et al., Screening for highly active beta-lactam antibiotics against Agrobacterium tumefaciens, Arch Microbiol, 181(4):331-6 (2004); Ogawa, Y., et al., Evaluation of 12 beta-lactam antibiotics for Agrobacterium-mediated transformation through in planta antibacterial activities and phytotoxicities, Plant Cell Rep, 23(10-11): 736-43 (2005). For these and other reasons, there is a need for the present invention.